Eye proteins have germ-killing power

When it comes to germ-busting power, the eyes have it, according to a
discovery by University of California, Berkeley (UC Berkeley) researchers that
could lead to new, inexpensive antimicrobial drugs.

A team of UC Berkeley vision scientists has found that small fragments of
keratin protein in the eye play a key role in warding off pathogens. The
researchers also put synthetic versions of these keratin fragments to the test
against an array of nasty pathogens. These synthetic molecules effectively
zapped bacteria that can lead to flesh-eating disease and strep throat (Streptococcus
pyogenes), diarrhea (Escherichia coli), staph infections (Staphylococcus
aureus), and cystic fibrosis lung infections (Pseudomonas aeruginosa).

The findings, to be published in the Journal of Clinical Investigation,
could lead to a powerful new weapon in the battle against disease-causing
invaders. These keratin fragments are relatively easy to manufacture, making
them good candidates for low-cost therapeutics, the study authors say.

"What's really exciting is that the keratins in our study are already in the
body, so we know that they are not toxic, and that they are biocompatible,"
says the study's principal investigator, Suzanne Fleiszig, a professor at UC
Berkeley's School of Optometry who specializes in infectious diseases and
microbiology. "The problem with small, naturally occurring, antimicrobial
molecules identified in previous research is that they were either toxic or
easily inactivated by concentrations of salt that are normally found in our
bodies."

These new small proteins in the study were derived from cytokeratin 6A, one
of the filament proteins that connect to form a mesh throughout the cytoplasm
of epithelial cells.

"We used to think that cytokeratins were primarily structural proteins, but
our study shows that these fragments of keratin also have microbe-fighting
capabilities," says study lead author Connie Tam, an assistant research scientist
in Fleiszig's laboratory. "Cytokeratin 6A can be found in the epithelial cells
of the human cornea as well as in skin, hair and nails. These are all areas of
the body that are constantly exposed to microbes, so it makes sense that they
would be part of the body's defense."

In a commentary published alongside the study, Michael Zasloff, professor of
surgery and pediatrics at Georgetown University’s School of Medicine, says
these "keratin-derived antimicrobial peptides appear to be exciting new
biocompatible candidates for development as human anti-infective therapeutics."

The researchers in Fleiszig's laboratory came upon cytokeratin 6A in their
efforts to solve the mystery behind the eye's remarkable resilience to
infection. They noticed that the surface of the eye, unlike other surfaces of
the body, did not have bacteria living on it, and that corneal tissue could
handily wipe out a barrage of pathogens in lab culture experiments.

"It is very difficult to infect the cornea of a healthy eye," says Fleiszig. "We've even used tissue paper to damage the eye's surface cells and then
plastered them with bacteria, and still had trouble getting bacteria to enter
the cornea. So we proposed that maybe there were antimicrobial factors that are
unique to the eye."

In the hunt for this mystery compound, the researchers cultured human
corneal epithelial cells and exposed them to the P. aeruginosa bacteria.
They used mass spectrometry to sort out which peptides were most active in
fighting off the bacteria. Cytokeratin 6A-derived peptides emerged the winners,
and surprisingly, peptide fragments as short as 10 amino acids were effective.

To confirm that they got the right protein, the researchers used
gene-silencing techniques to reduce the expression of cytokeratin 6A in the
cornea of mice. With a key defense disabled, the amount of bacteria that
adhered to the corneas increased fivefold.

Tests showed that cytokeratin 6A-derived fragments could quickly kill
bacteria in water and in a saline solution, showing that the salt contained in
human tears would not dilute the protein's effectiveness. Other experiments
indicated that cytokeratin 6A fragments prevented the bacteria from attacking
epithelial cells, and that the proteins cause bacterial membranes to leak,
killing the pathogen within minutes.

The researchers noted that further research could reveal numerous different
keratin fragments in the body’s innate defense system.

"Keratins may represent a novel class of antimicrobials with the potential
to be designed to selectively kill specific pathogens," says Tam.